Extract of R. miehei

ABSTRACT

Topical compositions comprising extracts of  Rhizomucor miehei  that are substantially devoid of acid-protease activity and their use in treating dermatologic conditions, including reducing the appearance of signs of skin aging.

FIELD OF INVENTION

The present invention is directed to novel topical compositionscomprising extracts of Rhizomucor miehei that are substantially devoidof acid-protease activity and their use in treating dermatologicconditions, including reducing the appearance of signs of skin aging.

BACKGROUND OF THE INVENTION

In order for milk to coagulate and eventually form cheese, enzymes mustbe added to breakdown the proteins that keep milk a liquid. Moreparticularly, when proteins are denatured or otherwise modified, milkloses its liquid structure and begins to coagulate. Rennets,milk-coagulating enzymes traditionally obtained from the abomasum (thefourth stomach of the calf) have long been used in cheese making. Themain enzyme in calf rennet is chymosin.

Calf-rennet, however, is expensive and has increasingly been replacedwith rennet derived from microorganisms. U.S. Pat. No. 4,526,792discloses the use of R. miehei as microbial rennet in the production ofcheese. (All documents cited are, in relevant, part, incorporated hereinby reference.) R. miehei does not contain chymosin, but instead acidproteases, which are similar in function to chymosin.

Among the commercially-available R. miehei extracts, several possesssome degree of enzymatic activity, principally from-acid proteases. Anextract substantially devoid of acid-protease activity can, however, beobtained by the removal of enzymatic activity by a number of well-knownbiochemical processes. The resulting non-enzymatic milieu issubstantially devoid of enzymatic activity. A number of methodswell-known to those skilled in the art to remove enzymatic activity areknown and include affinity gel column chromatography and subsequentelution of the adsorbed microbial rennet. See, e.g., Kobayashi, et al.,“Rapid isolation of microbial milk-clotting enzymes by N-acetyl-(orN-isobutyryl)-pepstatin-aminohexylagarose” Anal. Biochem., 122: 308-312(1982) (microbial rennet from R. miehei purified by use of affinity gelcolumn using N-acetylpepstatin as affinity ligand). Enzymes can also beseparated on affinity gel columns using Cibacron Blue F3GA (“CB”). See,e.g., Dean, et al., “Protein purification using immobilized triazinedyes,” J. Chromatogr., 165: 301-319 (1979) and Burgett, et al.,“Cibacron Blue F3GA affiriity chromatography”, Am. Lab., 9(5): 74, 78-83(1977) (describing separation of enzymes on CB columns, including forexample, kinases and nucleases). U.S. Pat. No. 4,743,551 describes theuse of a blue dye affinity ligand and elution of the adsorbed rennet toproduce a purified R. miehei rennet. A proteinaceous extract of R.miehei substantially devoid of acid-protease activity can also obtainedby other techniques well-known to those of skill in the art, includingthermal inactivation and molecular weight sieve.

There exists a delicate homeostatic balance between the rates ofsynthesis and degradation in the skin cells and underlying connectivetissues including collagen and elastin, the principal structuralproteins in mammalian skin. This balance allows the cells and tissues toregenerate as well as repair and replace damaged cells and tissuescaused by environmental stressors. Thus, in the case of collagen, bothendogenous and exogenous signals regulate the transcription of collagenmRNA and its subsequent translation into non-structural collagen.subunits known as procollagen. More particularly, procollagen undergoespost-translational modification, including hydroxylation of proline andlysine residues to hydroxyproline and hydroxylysine. Procollagen issusceptible to degradation by collagenases, including matrixmetalloproteinases. After being secreted through Golgi apparatus,procollagen is further processed into collagen via proteolytic removalof noncollagenous portions of the polypeptide (i.e., by proteases). Thecollagen molecules are then assembled into mature collagen fibrilswhich, in turn are cross-linked, and are more resistant tometalloproteases.

The visible signs of aging (e.g., fine lines and wrinkles) arecorrelated with a decrease in the level of collagen in the skin. This iscan be attributed both to decreased synthesis as well as increasedenzymatic degradation by collagenases, in particular Collagenase I alsoknown as Matrix Metalloprotease 1 (MMP1). The degradative activity ofMMP1 is regulated by the concentration of an endogenous proteaseinhibitor, Tissue Inhibitor of Matrix Metalloprotease-1 (TIMP1).

Prior art compositions have attempted to increase collagen synthesis inthe skin by a number of molecular mechanisms. See, e.g., U.S. Pat. No.6,846,812. KTTKS is a pentapeptide is derived from a fragment of theC-terminal portion of Collagen I. It has been studied in detail byKatayama et al., “A pentapeptide from type I procollagen promotesextracellular matrix production,” J. Biol. Chem., 268(14): 9941-9944(1993).

Another approach to reducing the appearance of the signs of aging hasbeen to enhance epidermal cell turnover by applying exfoliants. Twowidely-used classes of exfoliants well-known to those of skill in theart are acid proteases (e.g., Cathepsin D-like), see, e.g., U.S. Pat.Nos. 6,656,701 and 6,569,437, and hydroxycarboxylic acids (e.g.,alpha-hydroxy acids, such as glycolic acid).

In a poster presented at the February 2005 annual meeting of theAmerican Academy of Dermatology in New Orleans, Leyden et al. describedthe use of a botanical extract from R. miehei that has Cathepsin-D likeactivity to enhance epidermal cell renewal (i.e., exfoliation) andthereby improve the appearance of environmentally damaged skin.Suprisingly, the proteinaceous extract of R. miehei of the presentinvention that is substantially devoid of acid protease activity,reduces the appearance of the signs of aging by a primary mode of actionthat is not based on exfoliation.

Thus, there remains a long-felt but as yet unmet need to increaseCollagen I by upregulating the gene(s) that codes for the synthesis ofCollagen I, and/or decrease levels of MMP1, either by upregulating thegene(s) that codes for TIMP1 or downregulating the gene(s) that codesfor the expression of MMP1. These needs are met by the proteinaceousextract of the present invention. Surprisingly, the extract of R. mieheiof the present invention that is substantially devoid of acid proteaseactivity upregulates expression not only of the aforementioned genes,but also notably upregulates the expression of genes that code forfibronectin and vimentin (extracellular matrix glycoproteins involved incell adhesion, differentiation, and migration) as well asprocollagen-lysine 2-oxoglutarate 5-dioxygenase (an enzyme involved inthe crosslinking of procollagen to form mature bundled collagen fibers).

SUMMARY OF THE INVENTION

The present invention is directed to novel topical compositionscomprising extract of Rhizomucor miehei substantially devoid ofacid-protease activity that upregulates expression of the gene(s) thatcode for Collagen I thereby helping to reduce the appearance of thesigns of aging in a dermatologically-acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The topical composition of the present invention comprises extract ofRhizomucor miehei that is substantially devoid of acid protease activityin a dermatologically-acceptable carrier.

For purposes of the present application, “substantially devoid of acidprotease activity” means a composition in which the combined acidprotease(s) present have a combined acid protease specific activity ofless than about 500 HUT units/mg. In a preferred embodiment, thecombined acid protease specific activity is less than about 50 HUTunits/mg. Still more preferably, the combined acid protease specificactivity is less than about 10 HUT units/mg.

Combined acid protease activity is measured using the followingvariation on the method described in Food Chemicals CODEX, pp. 496-497(Washington, DC: National Academy Press, 1981): Hemoglobin substrate isprepared by mixing 2 g bovine hemoglobin into 80 ml distilled water. Thesolution is then titrated to pH 2 by adding, for example, phosphoricacid and/or citric acid. Additional distilled water is added for a totalvolume of 100 ml. The solution is separated into four equal portions.Each portion is titrated to the desired pH with 50% sodium hydroxide or50% hydrochloric acid. These final solutions are then heated at 30° C.for 20 minutes and are then filtered through glass wool. Trichloroaceticacid (“TCA” stock is prepared by dissolving TCA in distilled water for afinal concentration of 5% TCA.

For each sample for which proteolytic activity is to be measured,prepare tubes labeled “B” and “T”. In each tube place 4 ml of thehemoglobin solution and place at 37° C. such that the sample isprewarmed to 37° C. Into the “T” tube, add 100 μg enzyme solution, swirlgently and incubate at 37° C. for 20 minutes. Next add 10 ml TCA stocksolution to each tube. Into the “B” tube add an equal amount of theenzyme as added to the “T” tube. (This is the control for backgroundabsorbance.) Centrifuge each tube and filter each sample through asyringe filter and place the filtered sample into a quartz cuvette forreading the absorbance at 280 nm. The actual absorbance is determined bysubtracting the “T” sample's absorbance from the background. A standardcurve can be generated by measuring known quantities of protease.

One HUT unit of proteolytic activity is defined as that amount of enzymethat produces, in one minute under the specified conditions of theassay, a hydrolysate whose absorbance at 280 nm is the same as that of asolution containing 1.10 μg per ml of tyrosine in 0.006 N hydrochloricacid. HUT units per gram are determined by the following formula:HUT/g=(absorbance at 280 nm×V)/(0.0084×T×W)

where V is the final volume of the test solution,

T is the reaction time in minutes, and

W is the dry weight of the original enzyme sample used in the assay

Protein concentration is determined by a method known in the art, suchas, for example, the Bradford Assay which is described in Ausubel etal., (Eds.); Current Protocols in Molecular Biology (John Wiley & Sons,Inc., N.Y., 1994).

Rhizomucor miehei

R. miehei is an article of commerce available from a number of sourcesincluding Valley Research Inc. (South Bend, Ind.) and Novozymes(Bagsvaerd, Denmark).

R. miehei substantially devoid of acid protease activity is obtained bymolecular weight sieve, thermal inactivation and pepstatin-affinity gelchromatography.

In the present invention, R. miehei is present at a concentration lessthan about 500 HUT units/mg. In a preferred embodiment, the combinedacid protease specific activity is less than about 50 HUT units/mg.Still more preferably, the combined acid protease specific activity isless than about 10 HUT units/mg.

Dermatologically-Accepiable Carrier

The present invention further comprises a dermatologically-acceptablecarrier, one that is suitable for topical application to the keratinoustissue and is compatible with the dermatocosmetic active ingredientsdescribed below. The carrier can be in a wide variety of forms,including, but not limited to, oil-in-water emulsions, water-in-oilemulsions, water-in-silicone emulsions, silicone-in-water emulsions,water-in-oil-in-water, and oil-in-water-in-oil emulsions, andoil-in-water-in-silicone emulsions. Suitable surfactants includeanionic, cationic, amphoteric, zwitterionic and non-ionic, includingthose listed in U.S. Pat. No. 6,197,319.

The International Cosmetic Ingredient Dictionary and Handbook (10^(th)Edition, 2004), published by the Cosmetic, Toiletries & FragranceAssociation, describes a wide variety of non-limiting cosmetic anddermatopharmaceutical ingredients commonly used in the skin careindustry, which are suitable for use in combination with the R. mieheiextract of the present invention. Examples of these ingredients include:antioxidants, anti-inflammatory agents, anti-acne agents, antimicrobialagents, astringents, humectants, moisturizers, pH adjusters, skinbleaching/lightening agents, skin soothing/healing agents and agentsthat help decrease the appearance of signs of aging.

Non-limiting examples of anti-acne ingredients suitable for use incompositions of the present invention include: resorcinol, sulfur,salicylic acid, benzoyl peroxide, erythromycin, and zinc. Furtherexamples of suitable anti-acne actives are described in U.S. Pat. No.5,607,980.

Non-limiting examples of skin bleaching and lightening agents which maybe topically delivered in the present invention include: hydroquinone,kojic acid, glabradin, ascorbic acid, magnesium ascorbyl phosphate andascorbyl glucosamine.

Non-limiting examples of antioxidants/radical scavengers suitable foruse in compositions of the present invention include: ascorbic acid(vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic acidderivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbylphosphate, ascorbyl sorbate); tocopherol (vitamin E) and its esters,including tocopherol sorbate, tocopherol acetate; butylatedhydroxybenzoic acids and their salts;6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; gallic acid andits alkyl esters, especially propyl gallate; uric acid and its salts andalkyl esters; sorbic acid and its salts; lipoic acid; amines (e.g.,N,N-diethylhydroxylamine, amino-guanidine); sulfhydryl compounds (e.g.,glutathione); coenzyme Q10 and its analogues, including withoutlimitation, idebenone; dihydroxyfumaric acid and its salts; lycinepidolate; arginine pilolate; nordihydroguaiaretic acid; bioflavonoids;curcumin; lysine; 1-methionine; proline; superoxide dismutase;silymarin; tea extracts; Vitis vinifera (grape) skin/seed extracts;melanin; and Rosmarinus officinalis (rosemary) extracts.

Non-limiting examples of skin soothing and/or healing agents suitablefor use in the present invention include: allantoin, aloe vera and itsderivatives, betulinic acid, bisabolol, dipotassium glycyrrhizinate,oleonolic acid, panthenol and derivatives, pantothenic acid and itsderivatives, and ursolic acid.

Non-limiting examples of agents that help decrease the appearance ofsigns of aging, include muccopolysaccharides (including hyaluronicacid), aldosamines (including n-acetyl glucosamines) andbiologically-active short-chain peptides (e.g., tri-, tetra-, penta-,and hexapeptides, and mixtures thereo).

In a preferred embodiment of the present invention, one or more of thecosmetic ingredients are botanically-derived (e.g. extracts).

Preferred botanically-derived anti-bacterial agents include, but are notlimited to, extract of Laurus nobilis (bay laurel), extract of Larreadivaricata (chapparal), extract of Rosa canina (rose hips) and extractof Scutellaria galericulata (skullcap).

Preferred botanically-derived anti-fungal agents include, but are notlimited to, extract of Laurus nobilis (bay laurel), extract ofCommiphora myrrha (myrrh) and extract of Melaleuca alternifolia (teatree oil).

Preferred botanically-derived anti-inflammatory agents include, but arenot limited to, extract of Iris versicolor (blue flag), extract ofCalendula officinalis (calendula), extract of Chamomilla recrutita(chamomile), extract of Tussilago farfara (coltsfoot), extract ofSymphytum officinale (comfrey) leaves, extract of Tanacetum parthenium(feverfew), extract of Panax ginseng (ginseng), extract of Gynostemmapentaphyllum (southern ginseng), extract of Aesculus hippocastanum(horse chestnut), extract of Camellia oleifera (Japanese green tea),extract of Tilia cordata (linden tree), extract of Althea officinalis(marsh mallow), extract of Viola tricolor (pansy), extract of Menthapulegium (pennyroyal), extract of Vinca minor (periwinkle), extract ofChaenomeles japonica (quince) seed, extract of Anthemis nobilis (romanchamomile), extract of Valeriana officinalis (valerian) and extract ofViola odorata (violet).

Preferred botanically-derived agents that help decrease the appearanceof signs of aging include those that stimulate production of collagen.Non-limiting examples of these ingredients include asiatic acid,maddecassic acid and asiaticoside.

Preferred botanically-derived antioxidants include, but are not limitedto, extracts of Camellia oleifera (Japanese green tea), extracts ofVitis vinifera (grape) seed, extracts of Punica granatum (pomegranate),extracts of Citrus grandis (grapefruit), bioflavonoids, extracts ofPanax ginseng (ginseng), extracts of Gynostemma pentaphyllum (southernginseng), resveratrol, anthrocyanidines, monoterpenoids, diterpenoidsand triterpenoids,

Preferred botanically-derived astringent agents include, but are notlimited to, extract of Citrus medica limonum (lemon), extract of Citrusaurantifolia (lime), extract of Artium lappa (burdock), extract ofNasturtium officinale (watercress), extract of Hedera helix (ivy),extract of Hamamelis virginiana (witch hazel), extract of Myricacerifera (bayberry) rootbark, extract of Quercus alba (oak gall),extract of Echinacea purpurea (coneflower), extract of Echinaceaaugustifolia (native coneflower), extract of Eugenia caryophyllus (cloveoil), extract of Capsicum annum (cayenne pepper), extract of Menthapiperita (peppermint oil), and extract of Melaleuca altemifolia (teatree oil).

Preferred botanically-derived moisturizing agents include, but are notlimited to, pectin, disaccharides, oligosaccharides, and polysaccharidesextracted from Aloe barbedensis, algae, seaweed and sea grass.

Preferred botanically-derived skin bleaching/lightening agents include,but are not limited to, arbutin and glabradin.

The above-listed botanical extracts are commercially-available fromActive Organics LP (Lewisville, Tex.).

Sunscreen actives may also be used in combination with the R. mieheiextract of the present invention. These include the sunscreens currentlylisted by the US Food and Drug Administration in the Sunscreen DrugProducts for Over-The-Counter Human Use Final Monograph published in 64Federal Register pp. 27666-27693 (May 21, 1999). Other sunscreen activeingredients are accepted for use in countries outside the US and arealso considered to be within the scope of the present invention.

Dermatopharmaceutical ingredients that can be used in combination withthe R. miehei extract of the present invention are disclosed in U.S.Pat. No. 6,277,892, in Kerdel, et al, Dermatologic Therapeutics (2005),and in Hardman et al., Goodman & Gilman's: The Pharmacological Basis ofTherapeutics (10^(th) Edition, 2001). Further examples of cosmeticand/or dermatopharmaceutical ingredients which are suitable for use inthe delivery system of the present invention are disclosed in U.S. Pat.Nos. 6,492,326.

The following examples are further illustrative of the presentinvention. The components and specific ingredients are presented asbeing typical, and various modifications can be derived in view of theforegoing disclosure within the scope of the invention. In theseexamples, the proteinaceous extract of R. miehei that is substantiallydevoid of acid-protease activity is referred to by its tradenameActipeptide™ M from Active Organics, LP (Lewisville, Tex.). Allpercentages, ratios and proportions herein are by weight, unlessotherwise specified. All temperatures are in degrees Celsius unlessotherwise specified.

FORMULATION EXAMPLES

Toner Deionized Water 93.190% Methyl Gluceth-20 1.000% Potassium Sorbate0.100% Sodium Benzoate 0.100% Phenoxyethanol 0.600% Citric acid 0.010%Mucor Miehei Extract, 5.000% Butylene Glycol, and N-Acetylglucosamine(Actipeptide ™ M, Active Organics)Add ingredients sequentially in order listed. Mix until clear. Endprocessing.

Face Cream Part A Deionized Water 62.600% Magnesium Aluminum Silicate0.400% Xanthan Gum 0.150% Acrylates/C₁₀₋₃₀ Alkyl 0.750% AcrylateCrosspolymer

Part B Butylene Glycol 4.000% Disodium EDTA 0.050%

Part C Hydrogenated Lecithin 0.500% Caprylic/Capric Triglyceride 8.000%Simmondsia Chinensis (Jojoba) Seed Oil 5.000% Octyl Palmitate 4.000%Cetearyl Alcohol 2.000% PEG-8 Stearate 1.000% PEG-100 Stearate 0.800%

Part D Triethanolamine 99% 0.100%

Part E Aloe Barbadensis Leaf Juice 5.000% (Activera ® 10x, ActiveOrganics) Phenoxyethanol 0.500% Potassium Sorbate 0.100%Methylisothiazolinone 0.050% Mucor Miehei Extract, Butylene Glycol,5.000% and N-Acetylglucosamine (Actipeptide ™ M, Active Organics)Sprinkle Magnesium Aluminum Silicate, Xanthan Gum, Acrylates/C₁₀₋₃₀Alkyl Acrylate Crosspolymer into vortex of water. Mix and heat to 80° C.Add Part B to Part A, mix and hold the temperature at 80° C. In aseparate vessel, mix Part C and heat to 75° C., mix until clear. AddPart C to Parts A and B, mix for 10 minutes. Add Part D to Parts ABC.Mix for 15 minutes. Switch to sweep mixing. Cool batch to 45° C. In aseparate container, add ingredients in Part E. Mix until uniform. At 45°C., add Part E, to Parts ABCD. Mix and cool to 25° C. End processing.

Eye Cream Part A Deionized Water 57.650% Acrylates/C10-30 Alkyl 0.300%Acrylate Crosspolymer Panthenol 0.100% Potassium Sorbate 0.100% DisodiumEDTA 0.100% Allantoin 0.100

Part B Caprylic/Capric Triglyceride 2.000% Dimethicone 3.000%Butyrospermum Parkii (Shea Butter) 2.000% Carthamus Tinctorius(Safflower) Seed Oil 2.000% Cetearyl Alcohol 1.500% Dimethiconol 1.300%Steareth-2 1.000% Steareth-21 0.500% Cyclomethicone 5.000%

Part C Triethanolamine 0.250%

Part D Carbomer 940 2% Solution 10.000%

Part E Mucor Miehei Extract, Butylene Glycol, 5.000% andN-Acetylglucosamine (Actipeptide ™ M, Active Organics)

Part F Sodium Hyaluronate (Actimoist ® Bio 2, 2.000% Active Organics)Phenonip 1.000% Aloe Barbadensis Leaf Juice (Activera ® 10X, 5.000%Active Organics) Tocopherol 0.100%Sprinkle Acrylates/C₁₀₋₃₀ Alkyl Acrylate Crosspolymer into vortex ofwater. Mix and heat to 75° C. Mix and heat Part B to 70° C. Add Part Bto Part A, mix for 10 minutes. Add Part C. Mix for 10 minutes. Add PartD. Mix and cool to 45° C. At 45° C., add Parts E and F. Mix and cool to25° C. End processing.

Lipstick Part A Ricinus Communis (Castor) Seed Oil 24.37% OctylPalmitate 33.33% Petrolatum 10.84% Beeswax 3.33% Paraffin Wax 3.33%Euphorbia Cerifera (Candelilla) Wax 5.20% Ozokerite 3.00% CopemiciaCerifera (Carnauba) Wax 2.50% Simmondsia Chinensis (Jojoba) Seed Oil8.00% Propylparaben 0.10%

Part B Polyglyceryl-4 Isostearate 1.00% Mucor Miehei Extract, Butylene5.00% Glycol, and N-Acetylglucosamine (Actipeptide ™ M, Active Organics)Mix and heat Part A to 80° C. Pre-mix Part B; add to Part A. Mix andpour into container.

Face Mask Part A Deionized Water 59.960% Aloe Barbadensis Leaf Juice5.000% (Activera ™ 10X, Active Organics) Glycerin 4.000% Caffeine 0.100%Acacia Gum 0.300% Chromium Oxide Green 0.500% Titanium Dioxide 3.000%Methylparaben 0.200%

Part B Glyceryl Stearate 6.000% Simmondsia Chinensis (Jojoba) Seed Oil1.500% Tocopheryl Acetate 0.100% Propylparaben 0.100%

Part C Bentonite 11.000%

Part D Phenoxyethanol 0.500% Citric Acid 50% 2.100% Mucor MieheiExtract, Butylene Glycol, 5.000% and N-Acetylglucosamine (Actipeptide ™M, Active Organics)

Part E Essential Oil (Spearmint) 0.070% Essential Oil (Peppermint)0.070%Mix and heat Part A to 75° C. Mix and heat Part B to 75° C. HomogenizePart A, then add Part B continuing, mixing in the homogenizer for 5minutes. Start to cool. At 60° C., add Part C; mix well. Continuecooling. At 45° C., add Parts D and E. Mix and cool to 25° C. Endprocessing.

Moisturizing Shampoo Part A Deionized Water 46.680% Aloe BarbadensisLeaf Juice 10.000% (Activera ® 10X, Active Organics)

Part B Sodium C14-16 Olefin Sulfonate 18.000% Cocamidopropyl Betaine18.000% Glucamate DOE-120 1.000%

Part C Phenoxyethanol 0.300% Kathon CG 0.020% Sodium Chloride 25%Solution qs Butylene Glycol and Spiraea Ulmaria Extract 1.000%(Actiphyte ® Queen of Meadow Concentrate, Active Organics)

Part D Mucor Miehei Extract, Butylene Glycol, and 5.000%N-Acetylglucosamine (Actipeptide ™ M, Active Organics)Mix and heat Part A to 50° C. Add Part B to Part A; mix until clear. AddParts C and D to Parts A and B. Mix and cool to 25° C. End processing.

Moisturizing Conditioner Part A Deionized Water 64.670% Aloe BarbadensisLeaf Juice 5.000% (Activera ® 10X, Active Organics) Panthenol 0.200%

Part B Jojoba Oil 2.000% Behentrimonium Methosulfate, Cetearyl Alcohol4.000% Stearamidopropyl Dimethylamine 2.000% Cetearyl Alcohol 4.500%PEG-100 Stearate 0.880% Glyceryl Stearate 1.200%

Part C Water, Phenyl Trimethicone, Cyclomethicone, 10.000%Polysilicone-11, Lecithin (Actiprime ™ 100, Active Organics)

Part D Mucor Miehei Extract, Butylene Glycol, 5.000% andN-Acetylglucosamine (Actipeptide ™ M, Active Organics) Phenoxyethanol0.500% Methylisothiazolinone 0.050%Mix and heat Part A to 75° C. Mix and heat Part B to 75° C. Add Part Bto Part A. After mixing, add Part C and mix. Cool until 45° C., the addPart D. Mix and cool to 25° C. End processing.

Face Serum Part A Deionized Water 80.850% Keltrol RD 0.250% ButyleneGlycol 0.400%

Part B Water 0.600% Potassium Sorbate 0.100%

Part C Water, Algae Extract, and Aloe Barbadensis Leaf 5.0% Juice(Actisea ® 100, Active Organics)

Part D Aloe Barbadensis Leaf Juice (Activera ® 10x, Active Organics)5.000% Phenoxyethanol 0.600% Neolone 950 0.050% Mucor Miehei Extract,Butylene Glycol, and 5.000% N-Acetylglucosamine (Actipeptide ™ M, ActiveOrganics)

Part E Water 2.000% Allantoin  0.1% Disodium EDTA  0.05%Mix Part A. Add pre-dissolved Part B; mix until uniform. Add Part C; mixuntil uniform. Add Part D; mix well. Add pre-dissolved Part E, mix untiluniform. End processing.

Clinical Testing

Reduction in the appearance of signs of aging are documented using fourtypes of clinical studies well-known to persons of skill in the art: (i)skin hydration based on impedance measurements; (ii) skin firmness basedon ballistometry; (iii) wrinkle reduction via surface facial lineassessment; (iv) skin smoothness via replicas and digital imageanalysis. In each study, twelve female subjects, ages 40-60, with normaland/or normal to dry skin, exhibiting the signs of aging, apply thefollowing formula twice daily in an amount of 3mg/cm²: Part A Water70.40 Butylene Glycol 10.00 Part B Stearic Acid 2.50 Caprylic/CapricTriglyceride 3.00 Glyceryl Stearate 3.50 PEG-100 Stearate 2.00 CetearylAlcohol 1.00 Ceteareth-20 1.00 Dimethicone 0.60 Part C Phenoxyethanol0.72 Methylparaben 0.16 Ethylparaben 0.04 Propylparaben 0.02Butylparaben 0.04 Isobutylparaben 0.02 Part D Actipeptide ™ M 5.00The above formula is produced according to the following procedure: Mixand heat Part A to 75° C. Mix and heat Part B in a separate container to75° C. Add Part B to Part A. Mix well and cool batch to 45° C. Add PartC to batch while mixing. Add Part D. Mix and cool to 25° C.

The test subjects are not currently using Retin-A, or analogs,corticosteroids, benzoyl peroxide, and/or topical antibiotics and havenot used these products within the past six months. The subjectslikewise have not had facial peels or dermabrasion within the last year.Additionally, the subjects do not have psoriasis, eczema, or atopicdermatitis.

One week prior to entering the study, subjects discontinue using skincare products (e.g., moisturizer, sunscreen, liquid make-up). They arealso given a bar of soap (Neutrgena) with which to wash their entirefacial area as often as they choose. This skin equilibration periodhelps to ensure that all subjects are at a baseline value and help tominimize possible confounding from previous skin care regimens. On thefirst day after the one-week skin equilibration, all study participantsare given instructions on how to use the test products. Subjects areinstructed to cleanse only with the Neutrogena soap for the remainder ofthe eight-week study.

Skin moisture content is assessed via impedance measurements on thecheek area of subjects using a Novameter DPM. At the end of the one weekskin equilibration period, two separate measurements are taken on eachsubject and averaged. The baseline average of 95.8 indicates therelatively dry skin conditions of the subject. As summarized in thetable below, significant increases in skin moisture content of 16.5%,and 24.2% in skin moisture content are observed after two, four andeight weeks of treatment: Subject Baseline 2 weeks 4 weeks 8 weeks 1 90112 111 113 2 98 101 107 110 3 98 116 121 124 4 98 110 111 114 5 95 114116 119 6 90 100 104 121 7 93 122 124 122 8 94 115 114 118 9 97 121 127131 10  99 111 117 115 11  95 112 110 115 12  103 106 106 126 Average95.8 111.7 114.0 119.0 % Change 16.5 19.0 24.2

Skin firmness is assessed using a ballistometer (custom—Dermac Labs,Conn). As is known to those of skill in the art, a ballistometer drops apendulum on the skin surface and measures the resultant bouncingpattern. More particularly, the ratio of the height of the first andsecond rebound peaks are calculated. As summarized in the followingtable, after four and eight weeks of treatment, significant increases inskin firmness of 21.8% and 25.9% are found. Subject Baseline 2 weeks 4weeks 8 weeks 1 3.23 3.11 2.56 2.42 2 4.12 3.45 3.05 3.04 3 4.67 4.643.2 2.87 4 4.05 3.9 3.34 3.19 5 5.12 4.67 4.03 3.77 6 3.8 3.6 2.96 2.8 74.03 3.54 3.21 3.04 8 3.5 3.26 2.93 2.7 9 3.56 3.4 2.86 2.67 10  3.783.67 3.05 3.1 11  4.1 3.86 3.05 2.89 12  3.92 3.86 3.2 2.97 Average 3.993.75 3.12 2.96 % Change −6.1 −21.8 −25.9

Superfacial lines (SFLs) are assessed using the method of Packman, andGans, “Topical Moisturizer: Quantification of their effects onSuperficial Facial Lines,” J. Soc. Cosmet. Chem. 29: 79-90, (1978). Attwo weeks, four weeks and eight weeks, depth, shallowness and the numberof SFLs are scored within a defined area around the eye. Reductions of22.4%, 36.4% and 42.3% are observed after two, four and eight weeks.Subject Baseline 2 weeks 4 weeks 8 weeks 1 21 17 14 12 2 14 11 10 9.5 316.5 14 11.5 11 4 22 17 12.5 12 5 17.5 13 9 10 6 19 15 13 12 7 16 12 1110 8 18 12.5 10 8.5 9 19.5 16 13 11 10  22 18.5 14.5 12 11  18.5 12.5 1010 12  17 13 12 9.5 Average 18.42 14.29 11.71 10.63 % Change −22.4 −36.4−42.3

Changes in fine lines and wrinkles and skin texture are also measured bytaking Silflo Replicas of the left and right canthus areas. Adhesiverings are placed on each canthus area and dental silicon replicatingmaterial is poured inside the rings. As soon as the dries (2-3 minutes)it is removed, the replicas are labeled with the subject number anddate. Black and white photographs of the replicas are scanned andconverted into black and white pixels, where black pixels highlightlines and wrinkles. The total number white (non-wrinkle) and black(wrinkles and lines) pixels are compared to a predetermined thresholdvalue. Reductions in fine lines and wrinkles of 14.1%, 21%, and 30.1%are observed, respectively, at two, four and eight weeks. SubjectBaseline 2 weeks 4 weeks 8 weeks 1 10294 10203 9506 8324 2 13211 1104510405 9045 3 16237 12495 11450 10302 4 16203 12402 10890 9940 5 110349820 9340 7894 6 12405 10808 9937 8094 7 13002 11056 10485 10003 8 95799304 8765 6794 9 12200 10365 10038 8905 10  8567 8340 8244 7892 11 12345 10869 9450 8732 12  15230 12405 10203 9122 Average 12525.5810759.33 9892.75 8753.92 % Change −14.1 −21.0 −30.1

DNA Microarray Analysis

The effect of proteinaceous extract of R. miehei that is substantiallydevoid of acid-protease activity on the expression of certain genes areanalyzed using DNA microarrays as described below.

Cultured cells are grown in a 6-well plate until confluent usingappropriate culture conditions. Upon reaching confluency, three of thesix wells are treated with culture media supplemented with test materialat a concentration of 1%. The remaining three wells are treated withculture media alone and act as a control. After applying the testmaterial, the cells are incubated for 24 hours at 37±2° C. and 5±1% CO₂.At the end of the incubation period, the culture media is removed viaaspiration and the cells are washed once with cold phosphate bufferedsaline (“PBS”) using approximately 1 ml per well. After the wash, atrypsin/EDTA solution is added to the wells to detach the cells. Trypsinneutralizing solution is then added to the wells. The treated cells andthe untreated cells are pooled into separate 15 ml centrifuge tubes andpelleted by centrifuging at 1000 RPM at 4±2° C. After removing thesupernatant, the pelleted cells are lysed by adding 300 ml ofguanidinium thiocyanate lysis solution to each tube and then repeatedlydrawing and releasing the solution into the pipette until the cellpellet is dissolved. The cell lysates are stored at −75° C. until theRNA extraction process as described below is completed.

Alternatively, cultured tissues may be used. Tissue samples are removedfrom the shipping tray and placed into a 6-well plate containing 2.5-5.0ml of assay medium (37±2° C.). They are incubated for at least 24 hoursat 37±2° C. and 5±1% CO₂ After this initial incubation, the assay mediumis replaced with 2.5-5.0 ml of fresh medium (37±2° C.). 25-50 ml of testmaterial or phosphate buffered saline (negative control) are applieddirectly onto the surface of the tissue. The 6-well plates are thenincubated at 37±2° C. and 5±1% CO₂ for 24 hours. Thereafter, the tissuesamples are washed at least once with 100 ml of PBS and placed into a1.5 ml centrifuge tube containing 10-12 volumes of guanidiniumthiocyanate lysis solution. The tissues are minced with fine tippedscissors and homogenized until thoroughly disrupted. Afterhomogenization, the tissues are then centrifuged at 15,000 RPM for 10minutes. The supernatant is transferred to a new tube. The pellet(tissue debris) is discarded. The tissue homogenate is then stored at−75° C. until the RNA extraction process as described below iscompleted.

RNA Isolation

RNA isolation is performed using the RNAqueous Kit from Ambion Inc.(Austin, Texas). To the cell lysates or tissue homogenates preparedabove, an equal volume of 64% ethanol is added and the tubes arevortexed. Up to 700 ml of the mixture is transferred to a glass fiberfilter cartridge, which is loaded into a 1.5 ml collection tube and thecartridge is centrifuged for 1 minute at 14,000 RPM. The flow through isdiscarded. Any remaining mixture is loaded into the filter cartridge andthe centrifugation process is repeated until all of the mixture has beenprocessed. The filter is then washed to remove any residual cellulardebris from the RNA bound to the glass fibers by applying 700 ml of afirst wash solution (1 time) and 500 ml of a second wash solution (2times) to the filter cartridge and centrifuging at 14,000 RPM for 1minute to pass each wash through the cartridge. The flow through isdiscarded after each wash. After the final wash, one final spin isperformed without wash solution to remove any residual wash solution inthe filter cartridge. The RNA bound to the glass fibers within thecartridge is then be eluted by applying 30 ml of Tris-EDTA buffer (10 mMTris-HCl, 1 mM EDTA, preheated to 70-80° C., hereinbelow “TE buffer”) tothe cartridge and centrifuging the cartridge in a new collection tube at14,000 RPM for one minute. For samples prepared from cell lysates andsmall tissues, the elution process is repeated with an additional 30 mlof preheated TE buffer. For samples prepared from larger tissues (i.e.,full thickness tissues) the elution process is repeated two additionaltimes. After the RNA is eluted, RNA concentration is quantified using aRibogreen assay. RNA quality is assessed via gel electrophoresis.

RNA Concentration Assay

Ribogreen reagent is provided as a stock solution in DMSO. Prior to use,the reagent is diluted 2000 fold in TE buffer. The RNA assay requires200 ml of diluted Ribogreen reagent per sample to be tested and 1 ml ofthe reagent for the standards. Once prepared, the diluted reagent isstored protected from light. A series of RNA standards are prepared bydiluting purified ribosomal RNA derived from E. coli to the followingconcentrations: 2 mg/ml, 1 mg/ml, 200 ng/ml, 40 ng/ml and 0 ng/ml(blank). Prior to assaying, the RNA samples prepared above are diluted1000 fold in TE buffer. For the RNA assay, 100 ml of the diluted samplesor standards are transferred to the wells of a black 96-well plate. Thesamples and standards are assayed in duplicate. After thesamples/standards are added to the plate 100 ml of diluted Ribogreenassay reagent is added to the wells and the plate is gently mixed andallowed to incubate for 5-10 minutes protected from the light. Afterthis incubation, the plate is read with a fluorometer using anexcitation wavelength of 500 nm and an emission wavelength of 525 nm.

RNA Gel Electrophoresis

A 1% RNA gel is prepared by adding 0.3 g agarose to 21.6 mldiethylpyrocarbonate (DEPC) treated water. The agarose is dissolved byboiling the water in a microwave oven. After the solution is cooled toapproximately 55° C., 5.4 ml of formaldehyde and 3.0 ml 10×MOPS (0.2 MMOPS [pH 7.0], 20 mM sodium acetate, 10 mM EDTA, made in DEPC H₂O andfilter sterilized). After mixing, the agarose gel is cast in thehorizontal gel apparatus with loading slots placed on the side of thegel closest to the negative terminal. The gel is allowed to set for atleast 1 hour at room temperature. While the gel is setting, 175 ml of1×MOPS is prepared by diluting the 10× stock. After the gel is set, thecomb is removed and the buffer chamber of the gel apparatus is filledwith 150-175 ml 1×MOPS (enough buffer is added to cover the gel withapproximately 3 mm of buffer). The cover is placed on the apparatus, theelectrical leads are attached to the power source, and the empty gel isrun at 40 V (4 V/cm) for 5-10 minutes. While the gel is running, the RNAsamples are prepared by transferring approximately 1 mg of each sampleRNA to a 600 ml PCR tube. DEPC H₂O is used to bring the total volume ofall the samples to a common level and then 1-3 volumes of a gel-loadingbuffer (i.e. 5% glycerol, 1 mM EDTA, 0.025% bromophenol blue, 0.025%xylene cyanol FF, 20% formaldehyde, 50% formamide, 10 mg/ml ethidiumbromide) are added. The samples are denatured by placing them at 65-70°C. for 5-15 minutes and then placed on ice to cool. The samples are thencarefully loaded into the lanes (each loading slot can hold 10-15 ml ofsample, depending upon the thickness of the gel) and run on the gel at40 V for 1-3 hours. At the end of the run, the RNA is visualized byplacing the gel on a UV transilluminator. An RNA sample is used forsubsequent processing if both the 18S and 28S ribosomal bands areclearly visible and there is little or no staining below the 18S band.

mRNA Amplification

mRNA is amplified using the MessageAmp aRNA kit from Ambion Inc.

First Strand cDNA Synthesis: To start the first strand synthesis, 5 mgof total RNA for each sample are added to 600 ml PCR tubes and the totalvolume of liquid in the tube is adjusted to 12 ml with DEPC H₂O. To eachtube, 1 ml of T7 Oligo(dT) primer is added and the tube is incubated at70±2° C. for 10 minutes to denature the RNA and is then placed on ice toallow the primer to anneal to the poly A ends of the mRNA. Aftercooling, 2 ml of 10× first strand buffer, 1 ml of RNAse inhibitor and 4ml of dNTP mix is added to each tube, and the tube is placed at 42° C.As soon as the tube is heated, 1 ml of reverse transcriptase is addedand the tubes are returned to 42±2° C. for 2 hours. At the end of thetwo hours, the tubes are briefly centrifuged to collect all of the fluidat the bottom of the tube and then placed on ice.

Second Strand Synthesis and cDNA Purification: For the synthesis of thesecond strand of cDNA the following ingredients are added sequentiallyto the tubes: 63 ml DEPC H₂O, 10 ml 10× second strand buffer, 4 ml dNTPmix, 2 ml DNA Polymerase and 1 ml of RNAse H. The tube is mixed and thenincubated at 16±2° C. for 2 hours. Towards the end of the 2 hourincubation, a sufficient quantity of DEPC H₂O is warmed to 50±2° C., anda CDNA purification filter cartridge is equilibrated with 50 ml of cDNAbinding buffer (one cartridge per sample) for at least 5 minutes. Afterthe samples are finished incubating, 250 ml of cDNA binding buffer areadded to each tube and thoroughly mixed. The contents of the PCR tubeare then transferred to the cDNA purification filter cartridge. Thecartridge is then placed in a collection tube and centrifuged at 10,000RPM for 1 minute. The flow-through is discarded and 650 ml of cDNA washsolution is added to the cartridge. The cartridge is centrifuged again,the flow-through is discarded, and is then centrifuged one additionaltime to ensure that the wash buffer has been completely emptied from thefilter. The cDNA is eluted by applying 10 ml of preheated DEPC H₂O tothe filter and centrifuging the filter in a new collection tube at10,000 RPM for one minute. This elution is performed one additional timeto give a total volume of 16-18 ml of cDNA solution.

In Vitro Transcription to Synthesize aRNA and aRNA Purification

The in vitro transcription begins by adding the following to the cDNAsolution: 4 ml each of T7 ATP solution, T7 CTP solution, T7 GTPsolution, T7 UTP solution, 4 ml of 10× Reaction buffer, and 4 ml of T7enzyme mix. The tube is mixed and then incubated at 37±2° C. for 6-14hours. Towards the end of the incubation, a sufficient volume of ElutionSolution is warmed to 50-60° C. and an aRNA filter cartridge isequilibrated with 100 ml of aRNA binding buffer for at least 5 minutes.At the end of the incubation period, 350 ml of aRNA binding buffer isadded to the sample tubes and thoroughly mixed. An additional 250 ml ofabsolute ethanol is also added to each tube. The mixture is thentransferred to an aRNA filter cartridge; the cartridge is then insertedinto a collection tube and centrifuged at 10,000 RPM for 1 minute. Theflow-through is discarded and 650 ml of aRNA wash buffer is added to thecartridge followed by centrifuging at 10,000 RPM for one minute. Afterdiscarding the flow through, the cartridge is spun one final time toremove all traces of the wash buffer. The cartridge is then transferredto a new collection tube. 25 ml of pre-warmed Elution Solution is addedto the cartridge. The cartridge is incubated for 2 minutes at roomtemperature and then aRNA is eluted by centrifuging for 1 minute at10,000 RPM. This elution is performed one additional time to give atotal volume of 45-50 ml of aRNA solution. The final concentration ofthe aRNA is determined by the Ribogreen assay described above. Inaddition, the quality of the aRNA is checked via gel electrophoresis asdescribed above. An aRNA sample is used for subsequent processing if abroad band of RNA is observed.

Labeling and Purification of aRNA

aRNA is labeled with fluorescent dyes using the PerkinElmer ASAP RNALabeling Kit. Two tubes are prepared for the labeling process—for theuntreated sample Cy3 labeling (green), and for the treated sample Cy5labeling (red). To the Cy3 tube add 2 mg of aRNA prepared from theuntreated/control sample and add enough DEPC H₂O to bring the totalvolume up to 4 ml. To the Cy5 tube add 2 mg of aRNA prepared from thesample treated with the test material and add enough DEPC H₂O to bringthe total volume up to 4 ml. To both tubes, add 5 ml of ASAP labelingbuffer and 1 ml of the specific dye for the tube (Cy3 or Cy5). Incubatethe tubes for 15 minutes at 85±2° C. At the end of the 15 minutes, placethe tubes on ice to cool and then add 2.5 ml of ASAP stop solution toeach tube. The above proportions are sufficient for analyzing onemicroarray chip. If more chips are to be used then the labeling isincreased proportionately.

To purify the labeled aRNA, a microcon YM-30 filter column is insertedinto a collection tube and filled with 400 ml of TE buffer. The Cy3 andCy5 probes are combined (12.5 ml of each) and then added to the microconfilter and thoroughly mixed with the TE buffer. The filter iscentrifuged at 12,000 RPM for 8 minutes and the flow through isdiscarded. The column is washed twice with 400 ml of TE buffer,discarding the flow though each time. After the final wash, the filtercolumn is inverted, placed into a new collection tube and centrifuged at12,000 RPM for 2 minutes to collect the probe (the probe is concentratedin a volume of 2-30 ml of residual TE buffer).

Microarray Hybridization and Washing

For hybridization, 45 ml of 10× control target RNA (supplied withAgilent Technologies In Situ Hybridization Kit) is mixed with 160 ml ofDEPC water and 9 ml of 25× Agilent Fragmentation Buffer. This mixture isincubated at 60° C. for approximately 30 minutes in a hybridizationoven. At the end of the incubation, 225 ml of Agilent HybridizationBuffer is added along with the fluorescent aRNA probes prepared above.The mixture is then incubated at 70° C. for 5-10 minutes in a waterbath.During this incubation period, an Agilent SUREHYB hybridization chamberis prepared by inserting a glass gasket slide into the bottom half ofthe chamber. At then end of the incubation, the hybridization mixture(approximately 450 ml) is applied to the glass gasket slide and anAgilent Human 1A Oligo Microarray Chip is placed face down on top of thegasket such that the hybridization solution is sandwiched between theglass gasket slide and the microarray face of the chip. The top half ofthe chamber is attached and the connecting thumbscrew tightened. Afterverifying that there is good bubble formation in the chamber, it isplaced into the hybridization oven for approximately 17 hours (60° C.and rotating at 4 RPM). At then end of the hybridization period, themicroarray/glass gasket is removed from the SUREHYB chamber and placedin 50 ml of a first wash solution (room temperature, 6×SSC, 0.005%Triton X-102). After the gasket has fallen away from the microarray, thearray is transferred to 300 ml of fresh wash solution 1 on a magneticstir plate. The array is washed while the solution is mixed at mediumspeed for 10 minutes and is then transferred to 300 ml of wash solution2 (0.1×SSX, 0.005% Triton X-102, 4° C.) for 5 minutes. After the finalwash, the array is centrifuged at 500 RPM for 5 minutes until dry.

Microarray Scanning and Analysis

The microarrays are scanned with an Axon GenePix 4100A Scanner with thescanning resolution set to 10 mm and analyzed with GenePix Pro software.During the initial scan the PMT gains for the scanner are adjusted suchthat the Cy5/Cy3 image count ratios are between 0.88 and 1.12.

To derive the standard curve for the Ribogreen assay, the relativefluorescent units versus the known RNA concentrations in mg/ml for thestandards is plotted and subjected to regression analysis to establishthe line that best fits these data points. Mean RFU values for the testmaterials and untreated samples are then used to estimate the amount ofRNA present in each sample.

The level of gene expression is related to the fluorescence intensity ofthe probed gene marker on the microarray. Fluorescence measurementsbetween the the Cy3 and Cy5 probes are normalized. The total fluorescentsignal for both dyes is normalized with a correction factor such thatthe ratio of total intensities for both dyes equal to one.

Criteria for evaluating changes in gene expression are known to those ofordinary skill in the art and include the following: First, the ratio ofCy3/Cy5 (untreated/treated) fluorescence intensity is greater than 1.5or less than 0.66. (This relates to a change in gene expression of atleast +/−30%.) Second, the fluorescence intensity of the gene marker isgreater than the background intensity. Third, the gene feature isclearly marked specifically by the aRNA probes and is not due tonon-specific fluorescence. The first two criteria are filtered viacomputer analysis. The last criterion requires visual inspection of thearray.

Ratios of greater than about 1.3 are interpreted to indicate that a geneis upregulated by the treatment, whereas ratios of less than about 0.7are interpreted to indicate a downregulated gene. Thus, a ratio of 1.3,where the treated value is 130% of the untreated value, indicates a 30%increase in gene expression. Similarly, a ratio of 0.7 means that thetreated value was 70% of the untreated value, indicating a 30% decreasein gene expression.

The following results are observed after completing the micro-arrayanalysis as described above: Type 1 collagen, alpha 1 subunit 1.31 MMP10.495 Fibronectin 1.64 Vimentin 1.346 Procollagen-lysine 2-oxoglutarate5-dioxygenase 2.10

While the illustrative embodiments of the invention have been describedwith particularity, it will be understood that various othermodifications will be apparent to and can be readily made by thoseskilled in the art without departing from the spirit and scope of theinvention. Accordingly, it is not intended that the scope of the claimsappended hereto be limited to the examples and descriptions set forthhereinabove but rather that the claims be construed as encompassing allthe features of patentable novelty which reside in the presentinvention, including all features which would be treated as equivalentsthereof by those skilled in the art to which the invention pertains.claims

1. A dermatocosmetic composition comprising a therapeutically-effectiveamount of a proteinaceous extract of R. miehei substantially devoid ofacid-protease activity and a dermatologically-acceptable carrier.
 2. Thecomposition of claim 1 wherein the proteinaceous extract of R. mieheihas a combined acid protease specific activity of less than about 500HUT units/mg.
 3. The composition of claim 1 wherein the proteinaceousextract of R. miehei has a combined acid protease specific activity ofless than about 50 HUT units/mg.
 4. The composition of claim 1 whereinthe proteinaceous extract of R. miehei has a combined acid proteasespecific activity of less than about 10 HUT units/mg.
 5. A method fortreating or reducing the appearance of fine lines and wrinkles on theskin comprising administering to a person in need thereof thecomposition of claim
 1. 6. A method for treating or reducing theappearance of fine lines and wrinkles on the skin comprisingadministering to a person in need thereof the composition of claim
 2. 7.A method for treating or reducing the appearance of fine lines andwrinkles on the skin comprising administering to a person in needthereof the composition of claim
 3. 8. A method for treating or reducingthe appearance of fine lines and wrinkles on the skin comprisingadministering to a person in need thereof the composition of claim
 4. 9.A method for increasing levels of collagen in the skin comprisingadministering to a person in need thereof the composition of claim 1.10. A method for increasing levels of collagen in the skin comprisingadministering to a person in need thereof the composition of claim 2.11. A method for increasing levels of collagen in the skin comprisingadministering to a person in need thereof the composition of claim 3.12. A method for increasing levels of collagen in the skin comprisingadministering to a person in need thereof the composition of claim 4.13. A method for stimulating collagen synthesis comprising administeringto a person in need thereof the composition of claim
 1. 14. A method forincreasing the expression of one or more genes that code for collagensynthesis comprising administering to a person the composition ofclaim
 1. 15. A method for decreasing the expression of one or more genesthat code for collagenase comprising administering to a person thecomposition of claim
 1. 16. A method for increasing skin firmnesscomprising administering to a person in need thereof the composition ofclaim
 1. 17. A method for increasing skin firmness comprisingadministering to a person in need thereof the composition of claim 2.18. A method for increasing skin firmness comprising administering to aperson in need thereof the composition of claim
 3. 19. A method forincreasing skin firmness comprising administering to a person in needthereof the composition of claim
 4. 20. A method for increasing skinmoisture content comprising administering to a person in need thereofthe composition of claim
 1. 21. A method for increasing skin moisturecontent comprising administering to a person in need thereof thecomposition of claim
 2. 22. A method for increasing skin moisturecontent comprising administering to a person in need thereof thecomposition of claim
 3. 23. A method for increasing skin moisturecontent comprising administering to a person in need thereof thecomposition of claim 4.